Nel744b001kt

Paraffin sections (10 μm) were incubated overnight with primary antibodies, followed by 1h incubation with a fluorescent-dye-conjugated secondary antibody. RBPJ and N1ICD staining was performed using tyramide signal amplification (PerkinElmer NEL744B001KT). CBF:H2B-Venus expression was detected using anti-GFP antibody. Antibodies used in this study are: anti-RBPJ (CosmoBio 2ZRBP2, 1:50), anti-Troponin T (DSHB CT3, 1:20) anti-Cleaved Notch1 ICD (Cell Signaling Technology 2421S, 1:100), anti-GFP (Aves Labs GFP-1010, 1:400), and anti-Myosin Heavy Chain MF-20 (DHSB, 1:20). DAPI (Sigma-Aldrich D9542, 1:1000) and Isolectin B4 glycoprotein (ThermoFisher I32450, 1:100). Confocal images were obtained using Leica SP5 confocal fluorescence microscope.

Murine paraffin-embedded blocks were prepared from lungs fixed via the trachea with 10% buffered formalin at 20 cm H₂O. Serial sections were stained with anti-Ly6G clone 1A8 (ab25377, Abcam) and the following polyclonal antibodies: anti-SEMA3F (SAB2700501, Sigma-Aldrich), NRP1 (AF3870, R&D Systems), NRP2 (H300, Santa Cruz Biotechnology), or an isotype control following deparaffinization. Lung sections from patients with COPD undergoing resection for suspected lung tumor were stained with anti-SEMA3F, NRP2, or isotype control, and visualized with DAB or stained with anti-CD66b (555723, BD Biosciences), anti-NRP2 (HPA054974, Atlas Antibodies), and DAPI (422801, Sigma-Aldrich) with TSA plus system amplification (NEL744B001KT, Perkin Elmer) and autofluorescence quenching with TrueView (Vector, SP-8400).